Bifidobacterium breve, method for preparing selenium-rich preparation and / or small-particle-size high-activity nano-selenium particles and application of bifidobacterium breve

Abstract

The invention relates to a microbial fermentation technology, and discloses bifidobacterium breve, a method for preparing a selenium-rich preparation and / or small-particle-size high-activity nano-selenium particles, and application of the bifidobacterium breve. The strain name of the bifidobacterium breve is bifidobacterium breve ZZ409, and the preservation number of the bifidobacterium breve is CCTCC (China Center for Type Culture Collection) NO: M 20241663. The method for preparing the selenium-rich preparation and / or the nano-selenium particles comprises the following steps: carrying out anaerobic fermentation on the bifidobacterium breve and / or the microbial inoculum, and adding selenite in the anaerobic fermentation process. The process for preparing the nano-selenium particles from the bifidobacterium breve is simple, the extraction rate and the conversion efficiency are high, and the obtained nano-selenium particles can effectively kill tumor cells, have strong killing power to various pathogenic bacteria and are high in biological safety.

Description

Technical Field

[0001] The present invention relates to microbial fermentation technology, in particular to a Bifidobacterium breve, a method for preparing a selenium-enriched preparation and / or small-size high-activity nano-selenium particles and applications thereof. Background Art

[0002] Selenium (Se) is an essential trace element for the human body, playing a crucial role in maintaining human health and preventing the development of disease. Selenium can effectively reduce the incidence of various diseases, including vascular disease and cancer, and nano-selenium has significant therapeutic potential in antibacterial and anti-tumor applications. However, the vast majority of selenium in nature exists in an inorganic form, which is highly toxic and has low bioavailability. While nano-selenium can be synthesized using physical and chemical methods, these methods are associated with numerous challenges, such as high cost, complex procedures, and safety concerns.

[0003] Research has shown that some microorganisms can convert inorganic selenium into nano-selenium through their biological activities. Selenium-rich probiotics, primarily including selenium-enriched yeast, selenium-enriched Bifidobacterium, and selenium-enriched Lactobacillus, are capable of using inorganic selenium as a raw material and converting it into nano-selenium through their own biological activities. The production of nano-selenium using these selenium-rich microorganisms is low-cost, simple, and highly safe. The resulting nano-selenium can be widely used in health supplements and foods for special medical purposes, and possesses significant medicinal potential.

[0004] However, the efficiency of existing selenium-rich microorganisms in preparing nano-selenium is low and their functions are relatively simple, and their application value in the medical field is limited. Summary of the Invention

[0005] The purpose of the present invention is to overcome the problems of low efficiency, relatively single function and limited application value of microbial preparation of nano-selenium in the existing technology, and to provide a method and application of Bifidobacterium breve, preparing selenium-rich preparations and / or small-particle-size, high-activity nano-selenium particles. The process for preparing nano-selenium particles with Bifidobacterium breve is simple, and the extraction rate and conversion efficiency are high. The obtained nano-selenium particles can effectively kill tumor cells, have strong lethality to a variety of pathogens, and have high biosafety.

[0006] In order to achieve the above object, the present invention provides a first aspect of a Bifidobacterium breve strain named Bifidobacterium breve ZZ409 ( Bifidobacterium breve ZZ409), the deposit number is CCTCC NO: M 20241663.

[0007] A second aspect of the present invention provides a bacterial agent containing the aforementioned Bifidobacterium breve.

[0008] Preferably, the bacterial agent is a liquid bacterial agent or a solid bacterial agent.

[0009] Preferably, the bacterial agent is a liquid bacterial agent and the concentration of Bifidobacterium breve is 1×10 9 -1×10 11 CFU / mL.

[0010] The third aspect of the present invention provides the use of the aforementioned Bifidobacterium breve and the aforementioned bacterial agent in the preparation of selenium-enriched preparations and / or nano-selenium particles.

[0011] A fourth aspect of the present invention provides a method for preparing a selenium-enriched preparation and / or nano-selenium particles, the method comprising: subjecting the aforementioned Bifidobacterium breve and / or the aforementioned bacterial agent to anaerobic fermentation, and adding selenite during the anaerobic fermentation process.

[0012] Preferably, the final concentration of the added selenite is 10-60 μg / mL.

[0013] Preferably, the selenite is sodium selenite and / or potassium selenite.

[0014] Preferably, the conditions for anaerobic fermentation include: an inoculum size of 3×10 9 -3×10 10 CFU / mL, initial pH is 6-8, temperature is 30-40℃, and time is 20-40h.

[0015] Preferably, the selenite is added 8-12 hours after the start of the anaerobic fermentation.

[0016] Preferably, the culture medium for anaerobic fermentation contains peptone, beef powder, yeast powder, lactose, Tween 80, dipotassium hydrogen phosphate, sodium acetate, triammonium citrate, magnesium sulfate, and manganese sulfate.

[0017] Preferably, the culture medium for anaerobic fermentation contains 8-12 g / L of peptone, 4-6 g / L of beef powder, 3-5 g / L of yeast powder, 15-20 g / L of lactose, 0.5-1.5 mL / L of Tween 80, 1-3 g / L of dipotassium hydrogen phosphate, 4-6 g / L of sodium acetate, 1-3 g / L of triammonium citrate, 0.1-0.3 g / L of magnesium sulfate, and 0.03-0.08 g / L of manganese sulfate.

[0018] Preferably, the method further comprises: subjecting the fermentation liquid obtained by the anaerobic fermentation to cell disruption, solid-liquid separation, and washing to obtain the nano-selenium particles.

[0019] The fifth aspect of the present invention provides a selenium-rich preparation and / or nano-selenium particles prepared by the method described above.

[0020] Preferably, the particle size of the nano-selenium particles is 95-110 nm.

[0021] The sixth aspect of the present invention provides the use of the aforementioned Bifidobacterium breve, the aforementioned bacterial agent, the aforementioned selenium-rich preparation and / or nano-selenium particles in the preparation of functional products; wherein, the functional products have the function of treating colon cancer and / or resisting pathogenic bacteria.

[0022] Preferably, the pathogenic bacteria is selected from at least one of Staphylococcus aureus, Listeria monocytogenes, Escherichia coli and Shigella sonnei.

[0023] Through the above technical solution, the beneficial effects of the present invention are: The Bifidobacterium breve provided by the present invention can efficiently convert inorganic selenite into nano-selenium particles through its own growth metabolism, and the preparation process of the nano-selenium particles is simple and the extraction rate is high. The obtained nano-selenium particles can effectively kill tumor cells (colon cancer cells) and show strong lethality to multiple pathogenic bacteria (Staphylococcus aureus, Listeria monocytogenes, Escherichia coli and Shigella sonnei). It has high biosafety and can be used to prepare special medical foods, medicines, health products or auxiliary preparations for cancer treatment, etc., and has broad application prospects.

[0024] Biological Deposits The present invention provides Bifidobacterium breve ZZ409 ( Bifidobacterium breve ZZ409), was deposited on July 22, 2024 in the China Center for Type Culture Collection (address: Wuhan University, Wuchang District, Wuhan City, Hubei Province, Postal Code: 430072, the abbreviation of the depository is CCTCC), with the deposit number CCTCC NO: M 20241663. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a scanning electron microscope image of the selenium nanoparticles SeNPs in Example 2; Figure 2 is the EDS elemental analysis diagram of the nano-selenium particles SeNPs in Example 2; Figure 3 This is a graph showing the results of the investigation of factors affecting the particle size of nano-selenium particles SeNPs in Example 3; wherein A represents the effect of temperature on the particle size of SeNPs, B represents the effect of the initial pH of the culture medium on the particle size of SeNPs, C represents the effect of sodium selenite concentration on the particle size of SeNPs, D represents the effect of the time of sodium selenite addition on the particle size of SeNPs, and E represents the effect of the culture medium composition on the particle size of SeNPs; Ordinary one-way ANOVA followed by Tukey's multiple comparisons test was used to analyze significant differences, and different lowercase letters indicate significant differences; Figure 4Figure 4 shows the results of ROS measurement and cell viability staining after treatment of CT26 cells with nano-selenium particles SeNPs. A represents the ROS measurement (PC refers to the positive control, obtained by treating the cells with the positive control in the reactive oxygen species detection kit), and B represents the cell viability. Ordinary one-way ANOVA followed by Tukey's multiple comparisons test was used to analyze significant differences. Different lowercase letters indicate significant differences. Figure 5 1 is a graph showing the results of cell viability determination after treating Caco-2 and HepG2 cells with nano-selenium particles SeNPs in Example 4; wherein A represents Caco-2; B represents HepG2, and ns represents no significant difference. DETAILED DESCRIPTION

[0026] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0027] The first aspect of the present invention provides a Bifidobacterium breve strain named Bifidobacterium breve ZZ409 ( Bifidobacterium breve ZZ409), the deposit number is CCTCC NO: M 20241663.

[0028] The Bifidobacterium breve provided by the present invention is isolated from the feces of healthy infants (collected in Nanjing). The isolation of the Bifidobacterium breve can be carried out using conventional methods for isolating new strains in the art, for example, by picking a single colony using the plate streak method, repeatedly purifying to obtain a purified strain, extracting the genome of the purified strain, and performing PCR identification.

[0029] The breve bifidobacterium provided by the present invention can produce a large amount of viable cells of breve bifidobacterium after culture. The present invention has no particular limitation on the culture method, as long as the breve bifidobacterium can be proliferated in large quantities by the culture method. For example, the viable cells of breve bifidobacterium can be inoculated into a liquid culture medium, and anaerobically cultured at a temperature of 30-40°C to obtain a culture solution. Wherein, the liquid culture medium can be a culture medium conventionally used in the art, preferably containing: peptone 8-12g / L, beef powder 4-6g / L, yeast powder 3-5g / L, glucose 15-20g / L, Tween 80 0.5-1.5mL / L, dipotassium hydrogen phosphate 1-3g / L, sodium acetate 4-6g / L, triammonium citrate 1-3g / L, magnesium sulfate 0.1-0.3g / L, manganese sulfate 0.03-0.08g / L, for example, MRS liquid culture medium can be used.

[0030] The present invention can further separate the cells of Bifidobacterium breve from the above-mentioned culture medium. There is no particular limitation on the separation method, as long as the cells can be enriched from the culture medium. For example, it can be achieved by centrifugation and / or filtration. The conditions for the centrifugation and filtration can be conventional conditions in the art, which are well known to those skilled in the art and will not be described in detail here.

[0031] A second aspect of the present invention provides a bacterial agent containing the aforementioned Bifidobacterium breve.

[0032] According to the present invention, the bacterial agent preferably contains live cells of Bifidobacterium breve. There are no particular limitations on the dosage form of the bacterial agent. Depending on the intended use, the bacterial agent can be prepared into different dosage forms and contain corresponding excipients and other ingredients. For example, the bacterial agent can be a liquid agent (e.g., a bacterial solution of Bifidobacterium breve) or a solid agent (e.g., a powder prepared by drying Bifidobacterium breve). The addition of specific excipients to specific dosage forms of the bacterial agent is well known to those skilled in the art and will not be described in detail here.

[0033] In the present invention, there is no particular limitation on the concentration of Bifidobacterium breve in the bacterial agent, and a specific selection can be made according to specific circumstances. Further preferably, the bacterial agent is a liquid bacterial agent and the concentration of Bifidobacterium breve is 1×10 9 -1×10 11 CFU / mL, specifically 1×10 9 CFU / mL, 5×10 9 CFU / mL, 1×10 10 CFU / mL, 5×10 10 CFU / mL, 1×10 11 CFU / mL, or any value between the above two numbers.

[0034] The Bifidobacterium breve provided by the present invention can efficiently convert inorganic selenite into nano-selenium particles through its own growth and metabolism. The resulting nano-selenium particles can effectively kill tumor cells (colon cancer cells) and show strong lethality against multiple pathogens (Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, and Shigella sonnei). It also has high biosafety and can be used to prepare special medical foods, medicines, health products, or auxiliary preparations for cancer treatment. Based on this, in a third aspect, the present invention provides the use of the aforementioned Bifidobacterium breve and the aforementioned bacterial agent in the preparation of selenium-enriched preparations and / or nano-selenium particles.

[0035] In the present invention, the culture fluid obtained after culturing Bifidobacterium breve in a culture medium containing inorganic selenium can be directly used as a selenium-enriched preparation, or the selenium-enriched Bifidobacterium breve cells can be separated from the culture fluid and directly used as a selenium-enriched preparation, or the selenium-enriched Bifidobacterium breve cells can be compounded with other raw materials or auxiliary materials to form a selenium-enriched preparation. Nano-selenium particles can be obtained by extracting the selenium-enriched Bifidobacterium breve cells through cell disruption, centrifugation, washing, etc., and can also be used as a selenium-enriched preparation by themselves, or can be compounded with other raw materials or auxiliary materials to form a selenium-enriched preparation.

[0036] A fourth aspect of the present invention provides a method for preparing a selenium-enriched preparation and / or nano-selenium particles, the method comprising: subjecting the aforementioned Bifidobacterium breve and / or the aforementioned bacterial agent to anaerobic fermentation, and adding selenite during the anaerobic fermentation process.

[0037] From the perspective of resource conservation and environmental friendliness, the process for preparing selenium-rich preparations and / or nano-selenium particles using the Bifidobacterium breve provided by the present invention is simple and has low production cost, thus facilitating industrial production.

[0038] The Bifidobacterium breve provided by the present invention can convert inorganic selenium into nano-selenium particles through its own growth and metabolism, wherein the inorganic selenium is preferably selenite, and more preferably, the selenite is sodium selenite and / or potassium selenite. In this preferred embodiment, the conversion rate and production efficiency of the nano-selenium particles can be improved.

[0039] According to the present invention, preferably, the final concentration of the added selenite is 10-60 μg / mL, specifically 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, or any value between the two numbers. Under this preferred embodiment, not only can the conversion rate and generation efficiency of the nano-selenium particles be improved, but the particle size of the nano-selenium particles can also be reduced, thereby enhancing their anti-tumor and anti-pathogenic efficacy.

[0040] According to the present invention, anaerobic fermentation can be carried out using an anaerobic workstation. Preferably, the conditions for anaerobic fermentation include: an inoculation volume of 3×10 9- 3×10 10 CFU / mL, specifically 3×10 9 CFU / mL, 6×10 9 CFU / mL, 9×10 9 CFU / mL, 1.2×10 10 CFU / mL, 1.5×10 10 CFU / mL, 1.8×10 10 CFU / mL, 2.1×10 10 CFU / mL, 2.4×10 10 CFU / mL, 2.7×10 10 CFU / mL, 3×10 10 CFU / mL, or any value between the two numbers; the initial pH is 6-8, specifically 6, 6.5, 7, 7.5, 8, or any value between the two numbers; the temperature is 30-40°C, specifically 30°C, 32°C, 34°C, 36°C, 38°C, 40°C, or any value between the two numbers; the time is 20-40h, specifically 20h, 25h, 30h, 35h, 40h, or any value between the two numbers. Under this preferred embodiment, it is not only conducive to promoting the growth of Bifidobacterium breve and improving the generation efficiency of nano-selenium particles, but also can reduce the particle size of nano-selenium particles and enhance their anti-tumor and anti-pathogenic effects.

[0041] In the present invention, the selenite can be added at any time during the anaerobic fermentation. For example, it can be added directly to the initial culture medium so that it is present at the beginning of the fermentation; or it can be added after the fermentation starts and the Bifidobacterium breve is cultured to the logarithmic phase or the stable phase. Preferably, the selenite is added during the stable growth phase of Bifidobacterium breve after the anaerobic fermentation starts, which is conducive to further reducing the particle size of the nano-selenium particles and enhancing their anti-tumor and anti-pathogenic efficacy.

[0042] More preferably, when the total anaerobic fermentation time is 20-40 hours, the selenite is added 8-12 hours after the start of the anaerobic fermentation (during the stable growth period of the Bifidobacterium breve). Generally, the mother liquor of selenite is added to the anaerobic fermentation medium.

[0043] According to the present invention, the anaerobic fermentation can adopt common culture medium in this area, can provide required nutrients for the growth of Bifidobacterium breve, for example, carbon source can adopt glucose, lactose, sucrose etc., and nitrogen source can adopt peptone, yeast powder etc. Preferably, the culture medium of the anaerobic fermentation contains peptone, beef powder, yeast powder, lactose, Tween 80, dipotassium hydrogen phosphate, sodium acetate, ammonium citrate, magnesium sulfate, manganese sulfate. Further preferably, the culture medium of the anaerobic fermentation contains peptone 8-12g / L, beef powder 4-6g / L, yeast powder 3-5g / L, lactose 15-20g / L, Tween 80 0.5-1.5mL / L, dipotassium hydrogen phosphate 1-3g / L, sodium acetate 4-6g / L, ammonium citrate 1-3g / L, magnesium sulfate 0.1-0.3g / L, manganese sulfate 0.03-0.08g / L. This preferred embodiment is not only conducive to promoting the growth of Bifidobacterium breve and improving the generation efficiency of nano-selenium particles, but also can reduce the particle size of nano-selenium particles and enhance their anti-tumor and anti-pathogenic effects.

[0044] According to the present invention, Bifidobacterium breve is inoculated into the anaerobic fermentation medium in the form of a seed liquid; specifically, the seed liquid of Bifidobacterium breve can be obtained by inoculating Bifidobacterium breve into an MRS liquid medium and performing at least one seed culture. Further preferably, the concentration of Bifidobacterium breve in the seed liquid is 1×10 9 - 1×10 11 CFU / mL.

[0045] According to the present invention, preferably, the method further comprises: subjecting the fermentation liquid obtained by the anaerobic fermentation to cell disruption, solid-liquid separation, and washing to obtain the nano-selenium particles.

[0046] Exemplarily, the process of cell disruption, solid-liquid separation and washing includes: collecting bacteria from the fermentation broth obtained from anaerobic fermentation, washing with PBS (pH 7.4) at least 3 times and then resuspending in sterile water; adding lysozyme and incubating at a temperature of 35-40°C for 2-4 hours; then disrupting the mixture in an ultrasonic disruptor for 15-25 minutes to obtain a disrupted mixture containing cell fragments and nano-selenium particles; washing the disrupted mixture 3-5 times with Tris-HCl buffer (pH 8-9) containing 0.5-1.5% sodium dodecyl sulfate (SDS), and centrifuging to obtain a precipitate; washing the obtained precipitate and resuspending it in sterile water, and adding n-octanol to remove fat-soluble cell debris; vortexing the solution to mix thoroughly and then centrifuging, and the mixture after centrifugation is allowed to stand at a temperature of 0-10°C for 20-30 hours, and the bottom aqueous phase is washed with chloroform, 100% ethanol, 70% ethanol and deionized water in sequence, and the washed precipitate is dried to obtain purified nano-selenium particles SeNPs.

[0047] The fifth aspect of the present invention provides a selenium-rich preparation and / or nano-selenium particles prepared by the method described above.

[0048] According to the present invention, the particle size of the nano-selenium particles is 90-150 nm; more preferably, the particle size of the nano-selenium particles is 95-110 nm. This preferred embodiment is conducive to improving the anti-tumor and anti-pathogenic efficacy and biosafety of the nano-selenium particles.

[0049] The sixth aspect of the present invention provides the use of the aforementioned Bifidobacterium breve, the aforementioned bacterial agent, the aforementioned selenium-rich preparation and / or nano-selenium particles in the preparation of functional products; wherein, the functional products have the function of treating colon cancer and / or resisting pathogenic bacteria.

[0050] According to the present invention, preferably, the pathogenic bacteria is selected from at least one of Staphylococcus aureus, Listeria monocytogenes, Escherichia coli and Shigella sonnei.

[0051] In the present invention, the functional product can be a functional food, a special medical food, a medicine, a health product or an auxiliary preparation for cancer treatment, etc. The Bifidobacterium breve, bacterial agent, selenium-rich preparation and nano-selenium particles provided by the present invention can be used to prepare intermediates or active ingredients of functional products, or can be directly used as active ingredients of functional products.

[0052] The present invention will be described in detail below through examples.

[0053] In the following examples, Staphylococcus aureus Staphylococcus aureus ATCC 29213 (hereinafter referred to as SA), Listeria monocytogenes Listeria monocytogenes CMCC 54004 (hereinafter referred to as LM), Escherichia coli Escherichia coli ATCC 25922 (hereinafter referred to as EC), Shigella sonnei Shigella sonnei ATCC 25931 (hereafter referred to as SS) was purchased from the American Type Culture Collection (ATCC); sodium selenite (Na2SeO3, purity: 99%) was purchased from Tianjin Xiens Biochemical Technology Co., Ltd.

[0054] The human colon adenocarcinoma Caco-2 cell line was purchased from the American Type Culture Collection (ATCC) and cultured in Dulbecco's modified Eagle's medium (DMEM), which consists of DMEM high-glucose medium (with 100 IU / mL penicillin and 100 μg / mL streptomycin but without double-stranded antibodies; Keygen Biosciences, Jiangsu, China) supplemented with varying concentrations of fetal bovine serum. Caco-2 cells were cultured in DMEM high-glucose medium supplemented with 20% (v / v) premium South American fetal bovine serum (Mason Cell, Zhejiang, China).

[0055] Mouse colon cancer CT-26 cell line and human hepatocellular carcinoma HepG2 cell line were purchased from the American Type Culture Collection (ATCC) and cultured in Roswell Park Memorial Institute (RPMI)-1640 complete medium. RPMI-1640 complete medium consists of RPMI-1640 medium (with 100 IU / mL penicillin and 100 μg / mL streptomycin / without double-stranded antibodies, Keygen Biotechnology, Jiangsu, China) and 10% (v / v) fetal bovine serum (Gibco, Jiangsu, China).

[0056] The formula of MRS liquid medium is as follows: peptone 10 g / L, beef powder 5 g / L, yeast powder 4 g / L, glucose 2 g / L, Tween 80 1 mL / L, potassium dihydrogen phosphate 2 g / L, sodium acetate 5 g / L, ammonium citrate 2 g / L, magnesium sulfate 0.2 g / L, and manganese sulfate 0.05 g / L. The formula of MRS solid culture medium is MRS liquid culture medium with 15g / L agar added.

[0057] In the following examples, the anaerobic culture process was carried out using an anaerobic workstation (85% nitrogen, 10% hydrogen, 5% carbon dioxide; model: ELECTROTEK AW 400TG TWO GAS VERSION).

[0058] Unless otherwise specified, the remaining raw materials or reagents are conventional commercially available products.

[0059] Example 1 1.1 The present invention provides Bifidobacterium breve ZZ409 ( Bifidobacterium breve ZZ409) acquisition process This strain was isolated from the feces of healthy breastfed infants at Nanjing Gulou Hospital. Fresh feces were first collected and placed in preservation medium. After delivery to the laboratory, the feces were rapidly filtered and serially diluted, then plated onto MRS solid medium plates. The plates were incubated at 37°C in an anaerobic workstation (85% nitrogen, 10% hydrogen, 5% carbon dioxide; ELECTROTEK AW 400TGTWO GAS VERSION) for 48 hours. A single colony was then picked and inoculated into fresh MRS medium. The plates were then streaked and a single colony was picked. This procedure was repeated three times to obtain a pure strain. After the final single colony was picked and inoculated into fresh MRS liquid medium, the strain reached the logarithmic phase, the culture medium was harvested, the cells were centrifuged, and resuspended in sterile PBS supplemented with 25% glycerol for seed preservation.

[0060] 1.2 Physiological characteristics and molecular biological identification of strains The pure strain, grown on MRS solid-state medium, produces small, milky white colonies with distinct edges and a pleasant, sour aroma. Antimicrobial activity analysis confirmed that the organic acids secreted by the strain are the primary antimicrobial components, with lactic acid being the predominant.

[0061] The pure strain was cultured in MRS liquid medium, and the culture fluid was collected. The bacterial cells were collected by centrifugation and broken by adding bacterial lysis solution to extract DNA. PCR amplification was performed using the DNA as a template. The PCR products were collected and sent for testing. After sequencing data were spliced, sequence alignment was performed using BLAST in the NCBI database. Based on the alignment results, it was preliminarily determined to be Bifidobacterium breve (99.9~100.0%). The strain was subsequently named Bifidobacterium breve ZZ409 ( Bifidobacterium breve ZZ409), and was deposited in the China Center for Type Culture Collection (address: Room 211, China Center for Type Culture Collection, Wuhan University, Wuchang District, Wuhan City, Hubei Province, Postal Code: 430072, the abbreviation of the depository is CCTCC) on July 22, 2024, with the deposit number being CCTCC NO: M 20241663.

[0062] Example 2 2.1 Preparation of Se-BB bacterial suspension Bifidobacterium breve ZZ409 ( Bifidobacterium breve ZZ409 (hereinafter referred to as BB) was inoculated into MRS medium and placed in an anaerobic workstation (85% nitrogen, 10% hydrogen, 5% carbon dioxide; model ELECTROTEK AW 400TG TWOGAS VERSION) for 36 h at 37°C to obtain a concentration of approximately 2 × 10 10 CFU / mL of BB seed liquid.

[0063] The seed liquid was inoculated into fresh deoxygenated MRS liquid culture medium at an inoculum rate of 20% (V / V), and anaerobic fermentation was carried out at 37°C to obtain the selenium-enriched strain Se-BB bacterial liquid.

[0064] 2.2 Extraction and purification of selenium nanoparticles SeNPs The prepared selenium-enriched strain Se-BB was centrifuged (4°C, 10,000 rpm, 15 min) and the cells were collected. The cells were washed at least three times with PBS (pH 7.4) and resuspended in sterile water. 2 mL of lysozyme solution (100 mg / mL) was added and incubated at 37°C for 3 h. The mixture was then disrupted in an ultrasonic disruptor (XO-650D, Nanjing Xianou Instrument, China) for 20 min. The disrupted mixture containing cell debris and SeNPs particles was washed four times with 1.5 M Tris-HCl (pH 8.3) containing 1% sodium dodecyl sulfate (SDS) and centrifuged at 12,000 rpm for 10 min. The precipitate was washed and resuspended in 4 mL of sterile water, and 2 mL of n-octanol was added to remove fat-soluble cell debris. The solution was vortexed and thoroughly mixed and then centrifuged (4°C, 4300 rpm, 5 min). The centrifuged mixture was allowed to stand at 4°C for 24 h, and the bottom aqueous phase was washed with chloroform, 100% ethanol, 70% ethanol, and deionized water, in sequence. The washed precipitate was frozen at −80°C for 24 h and then freeze-dried in a vacuum freeze dryer (−60°C, 0 Pa) for 24 h to obtain the test sample.

[0065] The obtained samples were photographed under a scanning electron microscope (SU8010, HITACHI, Japan) and the particle size was calculated using the scale in the SEM image. Figure 1 It can be seen that a large number of nanospheres were produced outside the Se-BB cells. The results of EDS elemental analysis of the obtained samples are shown in Figure 2 , it can be known that it is selenium. Therefore, it can be determined that the sample to be tested is nano-selenium particles SeNPs.

[0066] Example 3 3.1 Effect of temperature on SeNPs particle size The BB seed solution prepared in Example 2.1 (concentration of about 2×10 10 CFU / mL), the bacterial cells were collected by centrifugation and resuspended in fresh MRS liquid culture medium containing 60 μg / mL sodium selenite, so that the inoculum size was about 3×10 10 CFU / mL, and then placed in anaerobic culture at 30℃, 37℃, and 42℃ for 24h to prepare Se-BB bacterial solution, and extracted nano-selenium particles SeNPs according to the method in Example 2.2 and took morphological pictures. The results are shown in Figure 3 In Figure A, the particle size of SeNPs converted by Bifidobacterium breve was the smallest when the culture temperature was 37°C.

[0067] 3.2 Effect of initial culture medium pH on SeNPs particle size The BB seed solution prepared in Example 2.1 (concentration of about 2×10 10 CFU / mL), the bacterial cells were collected by centrifugation and resuspended in fresh MRS liquid culture medium with pH values ​​of 6 / 7 / 8, respectively, so that the inoculum size was approximately 3×10 10 CFU / mL, then sodium selenite mother solution was added and the final concentration of sodium selenite was adjusted to 60 μg / mL, and Se-BB bacterial solution was prepared by anaerobically incubating at 37°C for 24 h. SeNPs were extracted according to the method in Example 2.2 and the morphology was photographed. The results are shown in FIG. Figure 3 In Figure B, it was found that the particle size of SeNPs converted by Bifidobacterium breve was the smallest when the initial pH was 7.

[0068] 3.3 Effect of sodium selenite addition time on SeNPs particle size The BB seed solution prepared in Example 2.1 (concentration of about 2×10 10 CFU / mL), the bacterial cells were collected by centrifugation and resuspended in fresh MRS liquid culture medium, so that the inoculum size was about 3×10 10 CFU / mL, and then placed at 37 ° C under the condition of anaerobically culturing Bifidobacterium breve to the logarithmic phase (about 6-8h after the start of fermentation) and the stable phase (about 8-12h after the start of fermentation), and then added sodium selenite mother liquor, and adjusted the final concentration of sodium selenite to 60μg / mL, and anaerobic culture was carried out for 24h to prepare Se-BB bacterial solution. Nano-selenium particles SeNPs were extracted according to the method in Example 2.2 and the morphology was photographed. The results are shown in FIG. Figure 3 Middle D, the particle size of SeNPs converted by Bifidobacterium breve was the smallest when sodium selenite was added during the stable period (8-12 h).

[0069] 3.4 Effect of sodium selenite concentration on SeNPs particle size The BB seed solution prepared in Example 2.1 (concentration of about 2×10 10 CFU / mL), the bacterial cells were collected by centrifugation and resuspended in fresh MRS liquid culture medium containing different concentrations of sodium selenite (the final concentrations of sodium selenite were 20, 40, 60, and 80 μg / mL, respectively), so that the inoculum size was about 3×10 10 CFU / mL, anaerobically cultured at 37 ° C for 24 h, Se-BB bacterial solution was prepared, and nano-selenium particles SeNPs were extracted according to the method in Example 2.2 and the morphology was photographed. The results are shown in Figure 3 In Figure C, it was found that with the increase of the final concentration of sodium selenite, the particle size of SeNPs showed a significant increasing trend. When the final concentration of sodium selenite was 20 μg / mL, the particle size of SeNPs transformed by Bifidobacterium breve was the smallest.

[0070] 3.5 Effect of culture medium components on SeNPs particle size The BB seed solution prepared in Example 2.1 (concentration of about 2×10 10 CFU / mL), the bacterial cells were collected by centrifugation and resuspended in fresh MRS culture medium containing different carbon sources (the final concentration of sodium selenite was 60 μg / mL, the carbon sources were glucose, sucrose, and lactose, each at 18 g / L; the other components were: peptone 10 g / L, beef powder 5 g / L, yeast powder 4 g / L, Tween 80 1 mL / L, potassium dihydrogen phosphate 2 g / L, sodium acetate 5 g / L, ammonium citrate tribasic 2 g / L, magnesium sulfate 0.2 g / L, and manganese sulfate 0.05 g / L), so that the inoculum size was approximately 3 × 10 10 CFU / mL, anaerobically cultured at 37 ° C for 24 h, Se-BB bacterial solution was prepared, and nano-selenium particles SeNPs were extracted according to the method in Example 2.2 and the morphology was photographed. The results are shown in Figure 3 In Figure E, it was found that the particle size of SeNPs cultured with lactose as the carbon source was significantly smaller than that of SeNPs cultured with sucrose and glucose as the carbon sources.

[0071] 3.6 Preparation of SeNPs with the smallest particle size SeNPs were prepared by taking the conditions with the smallest SeNPs particle size in the above single-factor experiments as parameters: the BB seed solution prepared in Example 2.1 (concentration of about 2×10 10 CFU / mL), the bacterial cells were collected by centrifugation and resuspended in fresh MRS culture medium (formula: peptone 10 g / L, beef powder 5 g / L, yeast powder 4 g / L, lactose 18 g / L, Tween 80 1 mL / L, potassium dihydrogen phosphate 2 g / L, sodium acetate 5 g / L, ammonium citrate tribasic 2 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, initial pH 7) to make the inoculum size about 3 × 10 10 CFU / mL, Bifidobacterium breve was then anaerobically cultured at 37°C until the stationary phase (approximately 6–8 hours after the start of fermentation). Sodium selenite stock solution was then added to adjust the final concentration to 20 μg / mL. Anaerobic culture was continued for 24 hours to produce a Se-BB bacterial solution. Selenium nanoparticles (SeNPs) were extracted according to the method described in Example 2.2, and their morphology was photographed and their particle size calculated. The prepared SeNPs had the smallest particle size, measuring 101.7 ± 5.23 nm.

[0072] Example 4 Determination of biological activity of nano-selenium particles SeNPs 4.1 Cellular ROS determination CT-26 cells were digested and centrifuged and resuspended in 1 mL of DMEM / RPMI-1640 complete medium. 20 μL was taken for cell counting. Based on the counting results, the cells were diluted to 2 × 10 5cells / mL, 1 mL per well was inoculated into a 12-well plate; the minimum-sized SeNPs prepared in Example 3.6 were washed three times with PBS and resuspended in DMEM / RPMI-1640 complete medium (SeNPs concentration was 80 mg / mL); When the CT-26 cells grew to a density of 70%, the culture medium was gently discarded and the cells were washed three times with PBS buffer. 1 mL of 80 mg / mL SeNPs solution was added to each well. The group without SeNPs was used as the negative control (NC), and the group treated with the positive control reagent in the reactive oxygen species detection kit was used as the positive control (PC). Three parallels were set up for each group. The cells were placed in an incubator (37°C, 5% CO2) and incubated for 24 h. Reactive oxygen species were measured using a reactive oxygen species detection kit (Biyuntian, Shanghai, China). The results are shown in Table 1. Figure 4 . Figure 4 Figure A shows that treating CT-26 cells with selenium nanoparticles SeNPs (101.7±5.23 nm) can induce cancer cells to produce more ROS.

[0073] 4.2 Evaluation of the killing effect of nano-selenium particles SeNPs on CT-26 CT-26 cells were digested and centrifuged and resuspended in 1 mL of RPMI-1640 complete medium. 20 μL was taken for cell counting. Based on the counting results, the cells were diluted to 2 × 10 5 cells / mL, 1 mL per well was inoculated into a 12-well plate; the minimum-sized SeNPs prepared in Example 3.6 were washed three times with PBS and resuspended in RPMI-1640 complete medium (SeNPs concentration was 80 mg / mL); When the CT-26 cells grew to a density of 70%, the culture medium was gently discarded and the cells were washed three times with PBS buffer. 1 mL of 80 mg / mL SeNPs solution was added to each well. The group without SeNPs was used as the negative control (NC). Three parallels were set up for each group. The cells were placed in an incubator (37°C, 5% CO2) and incubated for 24 h. The cells were stained for viability and death using the Calcein / PI cell viability and cytotoxicity detection kit (Biyuntian, Shanghai, China). The results are shown in Table 1. Figure 4 . Figure 4 B cell live-death staining showed that compared with the negative control group (NC group), almost all CT-26 cells were dead after SeNPs treatment.

[0074] The above experiments show that nano-selenium particles SeNPs can produce cytotoxic effects on cancer cells by increasing the production of ROS.

[0075] 4.3 In vitro safety evaluation of selenium nanoparticles (SeNPs) In vitro safety evaluation was performed using human colon adenocarcinoma cells Caco-2 and human liver cancer cells HepG2. Caco-2 / HepG2 cells were digested, centrifuged, and resuspended in 1 mL of DMEM / RPMI-1640 complete medium. 20 μL was taken for cell counting. Based on the count results, the cells were diluted to 2×10 with DMEM / RPMI-1640 complete medium. 5 cells / mL, 1 mL per well was inoculated into a 12-well plate; the minimum-sized SeNPs prepared in Example 3.6 were washed three times with PBS and resuspended in DMEM / RPMI-1640 complete medium (SeNPs concentration was 80 mg / mL); When Caco-2 / HepG2 cells grew to a density of 70%, the culture medium was gently discarded and the cells were washed three times with PBS buffer. 1 mL of 80 mg / mL SeNPs solution was added to each well. The treatment group without SeNPs was used as a negative control (NC). Three parallels were set up for each group. The cells were placed in an incubator (37°C, 5% CO2) for 24 h. The cells were stained for viability and death using the Calcein / PI cell viability and cytotoxicity detection kit (Beyotime, Shanghai, China). The results are shown in Table 1. Figure 5 The results showed that SeNPs did not affect the activity of Caco-2 and HepG2 cells.

[0076] 4.4 Antibacterial activity test of nano-selenium particles SeNPs The Oxford cup double-layer plate method was used to evaluate the inhibitory effect of equal amounts of nano-selenium particles SeNPs prepared in Example 3.6 (100 μL, 80 mg / mL) on Staphylococcus aureus (SA), Listeria monocytogenes (LM), Escherichia coli (EC), and Shigella sonnei (SS). The test bacterial cell size of SA, LM, EC, and SS was 1×10 4 CFU / mL, the inoculation volume was 100 μL, and the inhibition zone diameter (mm) was used as the evaluation standard. The results are shown in Table 1. It can be seen that the nano-selenium particles SeNPs provided by the present invention showed strong killing effect on Staphylococcus aureus, Listeria monocytogenes, Escherichia coli and Shigella sonnei.

[0077] Table 1 Diameters of inhibition zones of SeNPs against different pathogens

[0078] Note: Different lowercase letters in the same row indicate significant differences; the diameter of the Oxford cup is 7 mm.

[0079] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. A Bifidobacterium breve, characterized in that The strain is named Bifidobacterium breve ZZ409 ( Bifidobacterium breve ZZ409), the deposit number is CCTCC NO: M 20241663.

2. A bacterial agent, characterized in that The bacterial agent contains the Bifidobacterium breve according to claim 1.

3. The microbial agent according to claim 2, characterized in that The bacterial agent is a liquid bacterial agent or a solid bacterial agent; Preferably, the bacterial agent is a liquid bacterial agent and the concentration of Bifidobacterium breve is 1×10 9 -1×10 11 CFU / mL.

4. Use of the Bifidobacterium breve according to claim 1 or the bacterial agent according to claim 2 or 3 in the preparation of selenium-enriched preparations and / or nano-selenium particles.

5. A method for preparing a selenium-rich preparation and / or nano-selenium particles, characterized in that: The method comprises: subjecting the Bifidobacterium breve according to claim 1 and / or the bacterial agent according to claim 2 or 3 to anaerobic fermentation, and adding selenite during the anaerobic fermentation process.

6. The method according to claim 5, characterized in that The final concentration of selenite added is 10-60 μg / mL; Preferably, the selenite is sodium selenite and / or potassium selenite; Preferably, the conditions for anaerobic fermentation include: an inoculum size of 3×10 9 - 3×10 10 CFU / mL, initial pH 6-8, temperature 30-40°C, time 20-40h; Preferably, the selenite is added 8-12 hours after the start of the anaerobic fermentation.

7. The method according to claim 5 or 6, characterized in that The anaerobic fermentation culture medium contains peptone, beef powder, yeast powder, lactose, Tween 80, dipotassium hydrogen phosphate, sodium acetate, triammonium citrate, magnesium sulfate, and manganese sulfate; Preferably, the culture medium for anaerobic fermentation contains 8-12 g / L of peptone, 4-6 g / L of beef powder, 3-5 g / L of yeast powder, 15-20 g / L of lactose, 0.5-1.5 mL / L of Tween 80, 1-3 g / L of dipotassium hydrogen phosphate, 4-6 g / L of sodium acetate, 1-3 g / L of triammonium citrate, 0.1-0.3 g / L of magnesium sulfate, and 0.03-0.08 g / L of manganese sulfate.

8. The method according to claim 5 or 6, characterized in that The method further comprises: performing cell crushing, solid-liquid separation, and washing on the fermentation liquid obtained by the anaerobic fermentation to obtain the nano-selenium particles.

9. The selenium-rich preparation and / or nano-selenium particles prepared by the method according to any one of claims 5 to 8; Preferably, the particle size of the nano-selenium particles is 95-110 nm.

10. Use of the Bifidobacterium breve according to claim 1, the bacterial agent according to claim 2 or 3, the selenium-enriched preparation according to claim 9 and / or nano-selenium particles in the preparation of functional products; wherein, The functional product has the function of treating colon cancer and / or resisting pathogenic bacteria; Preferably, the pathogenic bacteria is selected from at least one of Staphylococcus aureus, Listeria monocytogenes, Escherichia coli and Shigella sonnei.

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